Oligonucleotides originating from sequences coding for the surface component of PTLV envelope proteins and their uses

ABSTRACT

The invention relates to the use of oligonucleotides from the nucleotide sequences coding for the amino-terminal region of the surface component (SU) of envelope proteins of PTLV viruses in order to perform methods of detecting every PTLV strain or PTLV-related viruses, e.g. for the detection of novel PTLV variants or viruses comprising sequences related to PTLV SUs. The invention also relates to primer pairs which are used to perform said detection methods and the novel PTLV variants thus detected.

A subject of the present invention is the oligonucleotides originatingfrom the nucleotide sequences coding for the amino-terminal region ofthe surface component of envelope proteins of the viruses of Tlymphomas/leukaemias in primates, grouped together under the designationPTLV, and their uses within the context of the detection of any strainof PTLV or related viral strains.

The present invention results from the identification by the Inventorsof peptide units of the SU which are suitable for the synthesis ofoligonucleotides which can be used for the detection and amplificationof pan-PTLV sequences comprising these units. The inventors havedeveloped a method allowing the amplification of such sequences, theircloning and sequencing. The present invention allows, in particular, thedetection of individual sequences present in a mixture of sequences ofdifferent types. Optimization for certain peptide units thus identifiedhas already allowed the characterization of PTLV variants which had notyet been described, as well as detecting PTLV sequences the presence ofwhich in the samples tested was not suspected. The generalizedapplication of the present invention will allow the detection andcharacterization either of novel sequences belonging to the SU of PTLV,or of sequences which are already known in new pathological or nonpathological contexts.

Research into sequences of human or primate retroviruses is of paramountimportance in numerous contexts. In a non-exhaustive manner, thisresearch concerns the screening of biological materials (productsderived from blood, for example), diagnosis (research into the etiologyof multiple syndromes covering leukaemias, degenerative diseases,autoimmune diseases, etc), epidemiological and anthropological studiesof different human groups, the sequencing of genomes (composition andpolymorphic retroviral markers of genomes), the screening of novelmedicaments (definition of new targets), etc.

In the case of PTLVs, we will demonstrate two examples of the problemsassociated with the detection of their sequences. In the first example,individuals, generally grouped under the term “seroindeterminate”,present an anti-HTLV immune response called “incomplete”, directedagainst certain antigens only of PTLVs, while no sequence correspondingto PTLVs can be amplified from blood samples of these patients. In thebest documented cases research into such sequences is carried out on theconserved regions of the gag, pol, env and tax genes. In the case of theenvelope gene, the amino-terminal part of the SU is excluded from thisapproach because of its variability. The amino-terminal region, of thesurface component (SU) of the envelopes of the retroviruses of human andnon-human primates of HTLV and STLV type (grouped together here underthe term PTLV) is in particular responsible for the recognition of thecell receptor or receptors of the envelope (Kim et al, 2000). To thisday, no method of amplification in this region which is directlyapplicable to the three types of PTLV (application called pan-PTLV) hasbeen described. Thus, in general only the amplification of units presentin the most conserved parts of the transmembrane component of theenvelope (TM) is considered. However, insofar as the variability of theSU is an essential element of the adaptive biology of the retrovirus,developing an approach based on its detection represents a particularlyuseful objective.

A subject of the present invention is the use of pairs of degenerateoligonucleotides in 5′ and 3′ orientation originating from thenucleotide sequences coding for the amino-terminal region of the surfacecomponent (SU) of the envelope proteins of the viruses of Tlymphomas/leukaemias in primates, grouped together under the designationPTLV, these viruses also being designated HTLV in man and STLV in themonkey, namely the region corresponding to the protein fragmentsdelimited on the N-terminal side by an amino acid situated betweenpositions 75 to 90, and on the C-terminal side by an amino acid situatedbetween positions 230 to 245 of the envelope proteins of the differentstrains of PTLVs, or of a virus carrying the sequences belonging to theSU of PTLVs,

for the implementation of processes for detecting any strain of PTLV,namely any strain belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, andSTLV-3, as well as any strain of virus belonging to PTLVs, namely anystrain the amino acid sequence of which is deduced from the nucleotidesequence coding for the amino-terminal region of the SU has an homologylevel of at least approximately 30% with the amino acid sequences codedby the corresponding nucleotide sequences in PTLVs, in particular fordetecting novel variants of PTLVs, or a virus, novel or not novel,comprising sequences belonging to the SU of PTLVs, if appropriate in newpathological contexts, said processes comprising an amplification stage,starting from a biological sample capable of containing PTLVs, and withthe abovementioned degenerate 5′ and 3′ oligonucleotides used asprimers, of the number of copies of nucleotide fragments delimited inposition 5′ by the degenerate oligonucleotide in 5′ orientation, and inposition 3′ by the degenerate oligonucleotide in 3′ orientation, and anidentification stage of the strain of PTLV contained in the biologicalsample from the abovementioned amplified nucleotide fragments.

A more particular subject of the invention is the abovementioned use ofpairs of degenerate oligonucleotides as defined above, characterized inthat said oligonucleotides are chosen from those comprisingapproximately 15 to approximately 30 nucleotides originating from thenucleotide sequences coding for protein fragments delimited on theN-terminal side by an amino acid situated between positions 75 to 90,and on the C-terminal side by an amino acid situated between positions230 to 245 of the envelope proteins of the different strains of PTLVs,such as the envelope protein of the MT-2 strain of HTLV-1 represented bySEQ ID NO: 43, or the NRA strain of HTLV-2 represented by SEQ ID NO: 45,or the strain of STLV-3 represented by SEQ ID NO: 47, said degenerateoligonucleotides comprising a mixture of oligonucleotides originatingfrom sequences coding for a determined region of approximately 5 to 10amino acids of the envelope proteins of the different strains of PTLV,and which differ from each other by the substitution of at least onenucleotide by another in a manner such that each oligonucleotide iscapable of coding for the abovementioned determined region originatingfrom the protein fragments of the envelope proteins of the differentstrains of PTLVs, such as the envelope protein of the MT-2 strain ofHTLV-1, or the NRA strain of HTLV-2, or the strain of STLV-3 which arementioned above.

A more particular subject of the invention is also the abovementioneduse of pairs of degenerate oligonucleotides as defined above, comprisingapproximately 15 to approximately 30 nucleotides originating fromnucleotide sequences coding for polypeptide fragments of approximately 5to approximately 10 amino acids originating from protein fragmentsdelimited by the amino acids situated at positions 80 to 245, and moreparticularly at positions 83 to 241, of the envelope protein of the MT-2strain of HTLV-1 (Gray et al., 1990, Virology, 177: 391-395; Genbankaccess No. M37747) represented by SEQ ID NO: 43.

Also, the invention relates more particularly to the abovementioned useof pairs of degenerate oligonucleotides as defined above, originatingfrom nucleotide sequences coding for polypeptide fragments 83-88,140-145, 222-228, and 237-241, of the envelope protein of the MT-2strain of HTLV-1, namely the following fragments:

83-YL/VFPHW-88

140-NFTQ/REV-145

222-NYS/TCI/MVC-228

237-WHVLY-241

Also, the invention relates more particularly to the abovementioned useof degenerate oligonucleotides in 5′ orientation originating from theDNA (+) strand coding for:

-   -   the polypeptide fragment 83-88 of the envelope protein of the        MT-2 strain of HTLV-1, said oligonucleotides being chosen from        those of following formula (I):

TAYBTNTTYCCNCAYTGG (I) SEQ ID NO: 5

in which:

Y represents C or T,

B represents C, G or T,

N represents A, C, G or T,

such as the 5′ oligonucleotide primers chosen from the following:

PTLVE5′83a TAYBTNTTYCCNCACTGG SEQ ID NO: 6 PTLVE5′83b TAYBTNTTYCCNCATTGGSEQ ID NO: 7

Y, B and N being as defined above,

-   -   or the polypeptide fragment 140-145 of the envelope protein of        the MT-2 strain of HTLV-1, said oligonucleotides being chosen        from those of following formula (II):

AAYTTYACNCARGARGT (II) SEQ ID NO: 8

in which:

Y represents C or T,

R represents A or G,

N represents A, C, G or T,

such as the 5′ oligonucleotide primers chosen from the following:

PTLVE5′140a AAYTTYACNCAAGAAGT SEQ ID NO: 9 PTLVE5′140b AAYTTYACNCAGGAAGTSEQ ID NO: 10 PTLVE5′140c AAYTTYACNCAAGAGGT SEQ ID NO: 11 PTLVE5′140dAAYTTYACNCAGGAGGT SEQ ID NO: 12

Y and N being as defined above.

Also, the invention relates more particularly to the abovementioned use,of degenerate oligonucleotides in 3′ orientation originating from theDNA (−) strand coding for:

-   -   the polypeptide fragment 140-145 of the envelope protein of the        MT-2 strain of HTLV-1, said oligonucleotides being chosen from        those of following formula (III):

NACYTCYTGNGTRAARTT (III) SEQ ID NO: 13

in which:

Y represents C or T,

R represents A or G,

N represents A, C, G or T,

such as the 3′ oligonucleotide primers chosen from the following:

PTLVE3′145a NACYTCYTGNGTAAAATT SEQ ID NO: 14 PTLVE3′145bNACYTCYTGNGTGAAATT SEQ ID NO: 15 PTLVE3′145c NACYTCYTGNGTAAAGTT SEQ IDNO: 16 PTLVE3′145d NACYTCYTGNGTGAAGTT SEQ ID NO: 17

Y and N being as defined above,

-   -   or the polypeptide fragment 222-228 of the envelope protein of        the MT-2 strain of HTLV-1, said oligonucleotides being chosen        from those of following formula (IV):

RMNACNATRCANSWRTARTT (IV) SEQ ID NO: 18

in which:

R represents A or G,

M represents A or C,

S represents C or G,

W represents A or T,

N represents A, C, G or T,

such as the 3′ oligonucleotide primers chosen from the following:

PTLVE3′228a RMNACNATRCANSAATAATT SEQ ID NO: 19 PTLVE3′228bRMNACNATRCANSAGTAATT SEQ ID NO: 20 PTLVE3′228c RMNACNATRCANSAATAGTT SEQID NO: 21 PTLVE3′228d RMNACNATRCANSAGTAGTT SEQ ID NO: 22 PTLVE3′228eRMNACNATRCANSTATAATT SEQ ID NO: 23 PTLVE3′228f RMNACNATRCANSTGTAATT SEQID NO: 24 PTLVE3′228g RMNACNATRCANSTATAGTT SEQ ID NO: 25 PTLVE3′228hRMNACNATRCANSTGTAGTT SEQ ID NO: 26

R, M, S, and N being as defined above,

-   -   or the polypeptide fragment 237-241 of the envelope protein of        the MT-2 strain of HTLV-1, said oligonucleotides being chosen        from those of following formula (V):

RTANARNACRTGCCA (V) SEQ ID NO: 27

in which:

R represents A or G,

N represents A, C, G or T,

such as the 3′ oligonucleotide primers chosen from the following:

PTLVE3′241a RTANARNACATGCCA PTLVE3′241b RTANARNACGTGCCA

R, and N being as defined above.

The invention also relates to the abovementioned use of oligonucleotidesas defined above, comprising at their 5′ end a sequence comprising arestriction site, such as the EcoRI site, of sequence GAATTC, or theBamHI site, of sequence GGATCC.

Therefore, the invention relates more particularly to the abovementioneduse of oligonucleotides as defined above, characterized in that the 5′oligonucleotides originating from the DNA (+) strand corresponding tothe polypeptide fragments 83-88 or 140-145 comprise at 5′ a sequenceGGAAGAATTC, and in that the 3′ oligonucleotides originating from the DNA(−) strand corresponding to the polypeptide fragments 140-145, 222-228,and 237-241 comprise at 5′ a sequence GGAAGGATCC.

A subject of the invention is also the abovementioned use ofoligonucleotides as defined above as probes, if appropriate labeled, forthe implementation of processes for detecting the abovementioned PTLVand related strains.

The invention also relates to the abovementioned use of oligonucleotidesas defined above, as pairs of nucleotide, primers for the implementationof polymerase chain reactions (PCR) for the detection of any strain ofPTLV, as well as any strain of virus comprising the sequences belongingto the SU of PTLVs.

A more particular subject of the invention is the abovementioned use ofpairs of primers chosen in such a way that:

-   -   the degenerate 5′ oligonucleotides correspond to a mixture of 5′        oligonucleotides originating from a same determined nucleotide        region comprising approximately 15 to approximately 30        nucleotides originating from the DNA (+) strand and coding for        the polypeptide fragments of approximately 5 to approximately 10        amino acids originating from protein fragments delimited on the        N-terminal side by an amino acid situated between positions 75        to 90, and on the C-terminal side by an amino acid situated        between positions 135 to 150 of the envelope proteins of the        different strains of PTLVs, in particular coding for the        polypeptide fragments of approximately 5 to approximately 10        amino acids originating from the protein fragment delimited on        the N-terminal side by the amino acid situated at position 83        and on the C-terminal side by the amino acid situated at        position 145 of the envelope protein of the MT-2 strain of        HTLV-1, said 5′ oligonucleotides being such that they differ        from each other by the substitution of at least one nucleotide        by another such that each oligonucleotide is capable of coding        for the abovementioned determined region originating from        protein fragments of the envelope proteins of different strains        of PTLVs, such as the envelope protein of the MT-2 strain of        HTLV-1, or the NRA strain of HTLV-2, or the strain of STLV-3        which are mentioned above,    -   the degenerate 3′ oligonucleotides correspond to a mixture of 3′        oligonucleotides originating from a same determined nucleotide        region comprising approximately 15 to approximately 30        nucleotides originating from the DNA (−) strand and coding for        the polypeptide fragments of approximately 5 to approximately 10        amino acids originating from protein fragments delimited on the        N-terminal side by an amino acid situated between positions 125        to 145, and on the C-terminal side by an amino acid situated        between positions 230 to 245 of the envelope proteins of the        different strains of PTLVs, in particular coding for the        polypeptide fragments of approximately 5 to approximately 10        amino acids originating from the protein fragment delimited on        the N-terminal side by the amino acid situated at position 140        and on the C-terminal side by the amino acid situated at        position 241 of the envelope protein of the MT-2 strain of        HTLV-1, said 3′ oligonucleotides being such that they differ        from each other by the substitution of at least one nucleotide        by another such that each oligonucleotide is capable of coding        for the abovementioned determined region originating from the        protein fragments of the envelope proteins of the different        strains of PTLVs, such as the envelope protein of the MT-2        strain of HTLV-1, or the NRA strain of HTLV-2, or the strain of        STLV-3 which are mentioned above,

it being understood that said abovementioned 5′ and 3′ primers cannot becomplementary to each other.

The invention relates more particularly to the abovementioned use ofpairs of degenerate oligonucleotides as defined above, characterized inthat the degenerate 5′ oligonucleotides are chosen from theabovementioned 5′ oligonucleotides of formulae (I) and (II), and in thatthe degenerate 3′ oligonucleotides are chosen from the abovementioned 3′oligonucleotides of formulae (III) to (V).

The invention relates more particularly to the abovementioned use ofpairs of primers as defined above, characterized in that the 5′ primeris chosen from the 5′ oligonucleotides originating from the DNA (+)strand corresponding to the polypeptide fragments 83-88 or 140-145defined above, such that the primers PTLVE 5′83 a and b and PTLVE 5′ 140a to d mentioned above, and in that the 3′ primer is chosen from the 3′oligonucleotides originating from the DNA (−) strand corresponding tothe polypeptide fragments 140-145, 222-228 or 237-241 defined above,such as the primers PTLVE 3′145 a to d, PTLV3′228 a to h, and PTLVE3′145 a to d, PTLV 3′228 a to h, and PTLVE 3′241 a and b mentionedabove.

A more particular subject of the invention is the abovementioned use ofpairs of degenerate oligonucleotides as defined above, saidoligonucleotides being chosen in such a way that they allow theamplification, starting from a biological sample capable of containingthe DNA of PTLV, of nucleotide sequences coding for the proteinfragments comprising a sequence delimited on the N-terminal side by theamino acid situated in position 89, and on the C-terminal side by theamino acid situated in position 139 of the envelope protein of the MT-2strain of HTLV-1 represented by SEQ ID NO: 43, or comprising ananalogous sequence comprised in the envelope protein of a strain of PTLVother than HTLV-1, such as the sequence delimited on the N-terminal sideby the amino acid situated in position 85, and on the C-terminal side bythe amino acid situated in position 135 of the envelope protein of theNRA strain of HTLV-2 represented by SEQ ID NO: 45, or the sequencedelimited on the N-terminal side by the amino acid situated in position88, and on the C-terminal side by the amino acid situated in position144 of the envelope protein of the strain of STLV-3 represented by SEQID NO: 47.

A more particular subject of the invention is also the abovementioneduse of pairs of degenerate oligonucleotides as defined above,characterized in that the degenerate 5′ oligonucleotides are chosen fromthe 5′ oligonucleotides of formula (I) mentioned above, and in that thedegenerate 3′ oligonucleotides are chosen from the 3′ oligonucleotidesof formulae (III) to (V) mentioned above.

The invention relates more particularly to the abovementioned use ofpairs of degenerate oligonucleotides as defined above, characterized inthat:

-   -   the degenerate 5′ oligonucleotides are those of following        formula (I):

PTLVE5′83b TAYBTNTTYCCNCATTGG SEQ ID NO: 5

Y, B and N being as defined above,

-   -   the degenerate 3′ oligonucleotides are those of following        formula (III):

PTLVE3′145a NACYTCYTGNGTAAAATT

Y and N being as defined above.

The invention also relates to the oligonucleotides as defined above, assuch.

Therefore, a more particular subject of the invention is theoligonucleotides as defined above, corresponding:

-   -   to the degenerate oligonucleotides in 5′ orientation originating        from the DNA(+) strand coding for:        -   the polypeptide fragment 83-88 of the envelope protein of            the MT-2 strain of HTLV-1, said oligonucleotides being            chosen from those of following formula (I):

TAYBTNTTYCCNCAYTGG (I) SEQ ID NO: 5

in which:

Y represents C or T,

B represents C, G or T,

N represents A, C, G or T,

such as the 5′ oligonucleotide primers chosen from the following:

PTLVE5′83a TAYBTNTTYCCNCACTGG SEQ ID NO: 6 PTLVE5′83b TAYBTNTTYCCNCATTGGSEQ ID NO: 7

Y, B and N being as defined above,

-   -   the polypeptide fragment 140-145 of the envelope protein of the        MT-2 strain of HTLV-1, said oligonucleotides being chosen from        those of following formula (II):

AAYTTYACNCARGARGT (II) SEQ ID NO: 8

in which:

Y represents C or T,

R represents A or G,

N represents A, C, G or T,

such as the 5′ oligonucleotide primers chosen from the following:

PTLVE5′140a AAYTTYACNCAAGAAGT SEQ ID NO: 9 PTLVE5′140b AAYTTYACNCAGGAAGTSEQ ID NO: 10 PTLVE5′140c AAYTTYACNCAAGAGGT SEQ ID NO: 11 PTLVE5′140dAAYTTYACNCAGGAGGT SEQ ID NO: 12

Y and N being as defined above,

-   -   to the degenerate oligonucleotides in 3′ orientation originating        from the DNA(−) strand coding for:        -   the polypeptide fragment 140-145 of the envelope protein of            the MT-2 strain of HTLV-1, said oligonucleotides being            chosen from those of following formula (III):

NACYTCYTGNGTRAARTT (III) SEQ ID NO: 13

in which:

Y represents C or T,

R represents A or G,

N represents A, C, G or T,

such as the 3′ oligonucleotide primers chosen from the followings:

PTLVE3′145a NACYTCYTGNGTAAAATT SEQ ID NO: 14 PTLVE3′145bNACYTCYTGNGTGAAATT SEQ ID NO: 15 PTLVE3′145c NACYTCYTGNGTAAAGTT SEQ IDNO: 16 PTLVE3′145d NACYTCYTGNGTGAAGTT SEQ ID NO: 17

Y and N being as defined above,

-   -   the polypeptide fragment 222-228 of the envelope protein of the        MT-2 strain of HTLV-1, said oligonucleotides being chosen from        those of following formula (IV):

RMNACNATRCANSWRTARTT (IV) SEQ ID NO: 18

in which:

R represents A or G,

M represents A or C,

S represents C or G,

W represents A or T,

N represents A, C, G or T,

such as the 3′ oligonucleotide primers chosen from the following:

PTLVE3′228a RMNACNATRCANSAATAATT SEQ ID NO: 19 PTLVE3′228bRMNACNATRCANSAGTAATT SEQ ID NO: 20 PTLVE3′228c RMNACNATRCANSAATAGTT SEQID NO: 21 PTLVE3′228d RMNACNATRCANSAGTAGTT SEQ ID NO: 22 PTLVE3′228eRMNACNATRCANSTATAATT SEQ ID NO: 23 PTLVE3′228f RMNACNATRCANSTGTAATT SEQID NO: 24 PTLVE3′228g RMNACNATRCANSTATAGTT SEQ ID NO: 25 PTLVE3′228hRMNACNATRCANSTGTAGTT SEQ ID NO: 26

R, M, S, and N being as defined above,

-   -   the polypeptide fragment 237-241 of the envelope protein of the        MT-2 strain of HTLV-1, said oligonucleotides being chosen from        those of following formula (V):

RTANARNACRTGCCA (V) SEQ ID NO: 27

in which:

R represents A or G,

N represents A, C, G or T.

such as the 3′ oligonucleotide primers chosen from the following:

PTLVE3′241a RTANARNACATGCCA SEQ ID NO: 28 PTLVE3′241b RTANARNACGTGCCASEQ ID NO: 29

R, and N being as defined above.

A subject of the invention is also a process for detecting any strain ofPTLV, namely any strain belonging to HTLV-1, HTLV-2, STLV-1, STLV-2, andSTLV-3, as well as any strain of virus comprising the sequencesbelonging to the SU of PTLVs, as defined above, characterized in that itcomprises:

-   -   the bringing into contact of a pair of degenerate 5′ and 3′        oligonucleotides as defined above, with the genomic DNA or        complementary DNA derived from RNA extracts of the content of a        biological sample (such as blood cells, bone marrow, biopsies,        in particular of the skin or other organs, or smears) capable of        containing PTLVs as defined above,    -   the amplification of DNA fragments coding for a fragment of the        envelope proteins of the different strains of PTLVs as defined        above,    -   the detection of the DNA fragments amplified during the previous        stage, this detection being able to be correlated to the        detection and if appropriate to the identification of PTLV as        defined above in said biological sample.

The invention also relates to a process for detecting any strain of PTLVas defined above, characterized in that the amplification stagecomprises the implementation of two amplification reactions, the secondreaction being carried out on a sample of products obtained within thecontext of the first reaction using the same 5′ oligonucleotides as inthe case of the first reaction, and 3′ oligonucleotides which aredifferent from those used in the first reaction, namely the so-called“nested” 3′ primers hybridizing with a region situated more upstream ofthe sequence coding for the SU than the primers 3′ used in the firstreaction.

A subject of the invention is also a process for detecting any strain ofPTLV as defined above, characterized in that it comprises:

-   -   a first gene amplification reaction carried out using pairs of        degenerate oligonucleotides chosen from the pairs:        -   oligonucleotides of formula (I)/oligonucleotides of formula            (IV), or        -   oligonucleotides of formula (I)/oligonucleotides of formula            (V), or        -   oligonucleotides of formula (II)/oligonucleotides of formula            (V),    -   and a second amplification stage of the number of copies of DNA        fragments obtained during the previous stage using pairs of        degenerate oligonucleotides chosen respectively from the pairs:        -   oligonucleotides of formula (I)/oligonucleotides of formula            (III), or        -   oligonucleotides of formula (I)/oligonucleotides of formula            (III or IV), or        -   oligonucleotides of formula (I)/oligonucleotides of formula            (IV),    -   the detection of the DNA fragments amplified during the previous        stage, this detection being able to be correlated to the        detection and if appropriate to the identification of PTLV in        the biological sample.

The invention also relates to a process for detecting any strain of PTLVas defined above, characterized in that it comprises:

-   -   a first gene amplification reaction carried out using pairs of        degenerate oligonucleotides chosen in such a way that:        -   the degenerate 5′ oligonucleotides are those of following            formula (I):

PTLVE5′83b TAYBTNTTYCCNCATTGG SEQ ID NO: 5

Y, B and N being as defined above,

-   -   the degenerate 3′ oligonucleotides are those of following        formula (V):

PTLVE3′241b RTANARNACGTGCCA SEQ ID NO: 29

R, and N being as defined above,

-   -   a second gene amplification reaction carried out using pairs of        degenerate oligonucleotides chosen in such a way that:        -   the degenerate 5′ oligonucleotides are those of following            formula (I):

PTLVE5′83b TAYBTNTTYCCNCATTGG SEQ ID NO: 5

Y, B and N being as defined above,

-   -   the degenerate 3′ oligonucleotides are those of following        formula (III):

PTLVE3′145a NACYTCYTGNGTAAAATT SEQ ID NO: 14

Y and N being as defined above.

The invention relates more particularly to a detection process asdefined above, characterized in that the amplification stage is carriedout under the following conditions:

-   -   denaturation at 94° C. for 5 minutes,    -   a first PCR reaction under so-called <<touch down>> conditions        carried out in a medium containing Taq polymerase or other DNA        polymerases which function at high temperature, this first PCR        reaction comprising:        -   15 <<touch down>> cycles together varying by the extension            temperature which reduces by 1° C. at each cycle comprising:            -   a denaturation stage at 94° C. for 15 seconds,            -   a combined stage of annealing and extension at a                temperature varying between 65° C. and 50° C. for 20                seconds,        -   30 standard cycles comprising:            -   a denaturation stage at 94° C. for 15 seconds,            -   an annealing stage at 50° C. for 30 seconds,            -   an extension stage at 72° C. for 30 seconds,    -   a second PCR reaction carried out on a sample of products        obtained in the context of the abovementioned first PCR reaction        using the same 5′ primer as in the case of the previous PCR        reaction, and a 3′ primer which is different to that used in the        previous PCR reaction, namely a so-called “nested” 3′ primer        hybridizing with a region situated more upstream of the sequence        coding for the SU than the 3′ primer used in the previous stage.

A more particular subject of the invention is a detection process asdefined above, characterized in that the detection stage, and ifappropriate the identification stage, is carried out under the followingconditions:

-   -   direct ligation of the fragments amplified during the        amplification stage in a plasmid such as pCR4-TOPO (Invitrogen),    -   transformation of bacteria with the abovementioned plasmid        comprising a marker gene such as a gene resistant to an        antibiotic, in particular to kanamycin,    -   subculturing the bacterial colonies (in particular between 10        and 100), culture, extraction of the DNA, and sequencing (in        particular using the universal primers T3 or T7 in the case of        the use of the vector pCR4-TOPO).

The invention also relates to a kit for the implementation of adetection process as defined above, characterized in that it comprises apair of abovementioned degenerate oligonucleotides, and, if appropriate,the reagents necessary for the implementation of the PCR amplificationreaction and for the detection of the amplified fragments.

A subject of the invention is also the application of the detectionprocess defined above for the diagnosis of pathologies linked to aninfection by a PTLV, or by a virus comprising sequences belonging to theSU of PTLV, in man or animals, such as hemopathies, autoimmune diseases,inflammatory diseases, degenerative diseases.

Therefore, the invention relates to any method of in vitro diagnosis ofthe abovementioned pathologies by implementation of a detection processdefined above, the detection of amplified DNA fragments being able to becorrelated to the diagnosis of said pathologies.

If appropriate, the in vitro diagnosis methods of the inventioncomprising an additional stage of identifying PTLV or viruses belongingto PTLVs present in the biological sample, by sequencing the amplifiedDNA fragments.

A subject of the invention is also the application of the detectionprocess defined above, with the screening and the identification ofnovel infectious agents in man or animals, and more particularly ofnovel strains (or variants) of a virus which can be classed in thePTLVs, or a virus comprising sequences belonging to the SU of PTLVs.

The abovementioned methods of screening and identification of novelinfectious agents are carried out by the implementation of a detectionprocess defined above and comprise an additional stage of identificationof novel variants of PTLV or of a related virus by sequencing theamplified DNA fragments.

The invention also relates to the application of the detection processdefined above by screening genes with a predisposition or a resistanceto the pathologies in man or animals linked to the presence of sequencesof PTLVs or of related sequences, or to an infection by a PTLV, such ashemopathies, autoimmune diseases, degenerative diseases.

A subject of the invention is also the application of the detectionprocess defined above, to the screening or the design of noveltherapeutic agents comprising entire or partial sequences of theenvelope proteins of novel variants of PTLV thus detected.

The invention also relates to the application of the detection processas defined above, to the screening or the design of novel cell therapyvectors using the tropism proprieties of entire or partial sequences ofthe envelope proteins of novel variants of PTLV thus detected.

A subject of the invention is also the variants of type HTLV-1 asobtained by implementation of a detection process defined above,corresponding:

-   -   to the variant, the envelope protein of which is such that it        comprises the following peptide sequence SEQ ID NO: 31:

I K K P N P  N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C PY T G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the arginine (R) residue in position 94,and the serine (S) residue in position 101, are replaced respectively bya proline (P) residue and a leucine (L) residue indicated in bold andunderlined,

and the nucleotide sequence of which coding for its envelope protein issuch that it comprises the following sequence SEQ ID NO: 30:

ATT AAA AAG CCA AAC C C A AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which G inposition 281, C in position 302, and G in position 333, are replacedrespectively by C, T, and A indicated in bold and underlined.

-   -   to the variant, the envelope protein of which is such that it        comprises the following peptide sequence SEQ ID NO: 33:

V  K K P N R N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C PY T G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the isoleucine (I) residue in position89, and the serine (S) residue in position 101, are replacedrespectively by a valine (V) residue and a leucine (L) residue indicatedin bold and underlined,

and the nucleotide sequence of which coding for its envelope protein issuch that it comprises the following sequence SEQ ID NO: 32:

G TT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which A inposition 266, C in position 302, and G in position 333, are replacedrespectively by G, T, and A indicated in bold and underlined,

-   -   to the variant, the envelope protein of which is such that it        comprises the following peptide sequence SEQ ID NO: 35:

I K K P N R N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C P YT G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the serine (S) residue in position 101,is replaced by a leucine (L) residue indicated in bold and underlined,

and the nucleotide sequence of which coding for its envelope protein issuch that it comprises the following sequence SEQ ID NO: 34:

ATT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CA A  CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which C inposition 302, G in position 333, and G in position 408, are replacedrespectively by T, A, and A indicated in bold and underlined,

-   -   to the variant, the envelope protein of which is such that it        comprises the following peptide sequence SEQ ID NO: 37:

I K K P N R N G G G Y Y L A S Y S D P C S L K C P Y L G C Q S W T C P YT G P  V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the alanine (A) residue in position 127,is replaced by a proline (P) residue indicated in bold and underlined,

and the nucleotide sequence of which coding for its envelope protein issuch that it comprises the following sequence SEQ ID NO: 36:

GTT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT TTA GCC TCT TAT TCA GACCCT TGT TCC TTA AAA TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGC CCC TATACA GGA C CC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which G inposition 379, is replaced by C indicated in bold and underlined,

-   -   to the variant, the envelope protein of which is such that it        comprises the following peptide sequence SEQ ID NO: 39:

I K K P N R N G G G Y H  S A S Y S D P C S L K C P Y L G C Q S W T C P YA  G A V S S P Y W K F Q Q D V N F T Q E V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 145 of the envelope protein of theMT-2 strain of HTLV-1, in which the tyrosine (Y) residue in position100, and the threonine (T) residue in position 125, are replacedrespectively by a histidine (H) residue and an alanine (A) residueindicated in bold and underlined,

and the nucleotide sequence of which coding for its envelope protein issuch that it comprises the following sequence SEQ ID NO: 38:

ATT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT C AT TCA GCC TCT TAT TCA GACCCT TGT TCC TTA AAG TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGC CCC TATG CA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC AAT TTT ACC  CA G  GAA GT A

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 435 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which T inposition 298, A in position 373, T in position 426, A in position 429,and T in position 435, are replaced respectively by C, G, C, G, and Aindicated in bold and underlined.

A subject of the invention is also the variant of type HTLV-2 asobtained by implementation of a detection process defined above,characterized in that:

-   -   its envelope protein is such that it comprises the following        peptide sequence SEQ ID NO: 41:

I R  K P N R Q G L G Y Y S P S Y N D P C S L Q C P Y L G S  Q S W T C PY T A  P V S T  P S W N  F H S D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 85 to 135 of the envelope protein of theprototype NRA strain of HTLV-2 (described by Lee et al., 1993. Virology196, 57-69; Genbank access No. L20734.1), in which the followingresidues: lysine (K) in position 86, cysteine (C) in position 113,glycine (G) in position 122, serine (S) in position 126, and lysine (K)in position 130, are replaced respectively by the following residues:arginine (R), serine (S), alanine (A), threonine (T), and asparagine (N)indicated in bold and underlined,

-   -   the nucleotide sequence coding for its envelope protein is such        that it comprises the following sequence SEQ ID NO: 40:

ATA A GA  AAG CCA AA C  AGA CAG GGC CTA GG G  TAC TAC TCG CC T  TCC TA C AAT GAC CCT TGC TCG CTA CAA TGC CCC TAC TT G  GGC T C C CAA TCA TGG ACATGC CCA TAC ACG G C C CCC GTC TCC A C T CCA TCC TGG AA T  TTT CA T  TCAGAT GTA

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 253 to 405 of the sequence coding forthe envelope protein of the NRA strain of HTLV-2, in which A in position257, G in position 258, T in position 267, A in position 282, C inposition 294, T in position 300, A in position 333, G in position 338, Gin position 365, G in position 377, G in position 390, and C in position396, are replaced respectively by G, A, C, G, T, C, G, C, C, C, T, and Tindicated in bold and underlined.

The invention also relates to the polypeptides delimited on theN-terminal side by an amino acid situated between positions 75 to 90,and on the C-terminal side by an amino acid situated between positions230 to 245 of the envelope proteins of the different strains of PTLVs,such as the envelope protein of the MT-2 strain of HTLV-1 represented bySEQ ID NO: 43, or the NRA strain of HTLV-2 represented by SEQ ID NO: 45,or the strain of STLV-3 represented by SEQ ID NO: 47, or a viruscarrying the sequences belonging to the SUs of PTLVs, or delimited onthe N-terminal side by an amino acid situated between positions 75 to90, and on the C-terminal side by an amino acid situated betweenpositions 135 to 150 of said envelope proteins of the different strainsof PTLVs.

A subject of the invention is also the polypeptides defined above,chosen from:

-   -   the polypeptide delimited on the N-terminal side by the amino        acid situated at position 83 or 89, and on the C-terminal side        by the amino acid situated at position 139 or 145, of the        envelope protein of the MT-2 strain of HTLV-1 represented by SEQ        ID NO: 43,    -   the polypeptide delimited on the N-terminal side by the amino        acid situated at position 79 or 85, and on the C-terminal side        by the amino acid situated at position 135 or 141, of the        envelope protein of the NRA strain of HTLV-2 represented by SEQ        ID NO: 45,    -   the polypeptide delimited on the N-terminal side by the amino        acid situated at position 82 or 88, and on the C-terminal side        by the amino acid situated at position 138 or 144, of the        envelope protein of the strain of STLV-3 represented by SEQ ID        NO: 47.

The invention relates also to the polypeptides coded by the DNAfragments amplified within the context of the detection process definedabove, of variants of type HTLV-1 to HTLV-2 mentioned above,characterized in that they comprise the following peptide sequences:

-   -   polypeptide 1 (SEQ ID NO: 31):

I K K P N P  N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C PY T G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the arginine (R) residue in position 94,and the serine (S) residue in position 101, are replaced respectively bya proline (P) residue and a leucine (L) residue indicated in bold andunderlined,

-   -   polypeptide 2 (SEQ ID NO: 33):

V  K K P N R N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C PY T G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the isoleucine (I) residue in position89, and the serine (S) residue in position 101, are replacedrespectively by a valine (V) residue and a leucine (L) residue indicatedin bold and underlined,

-   -   polypeptide 3 (SEQ ID NO: 35):

I K K P N R N G G G Y Y L  A S Y S D P C S L K C P Y L G C Q S W T C P YT G A V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the serine (S) residue in position 101,is replaced by a leucine (L) residue indicated in bold and underlined,

-   -   polypeptide 4 (SEQ ID NO: 37):

I K K P N R N G G G Y Y L A S Y S D P C S L K C P Y L G C Q S W T C P YT G P  V S S P Y W K F Q Q D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 139 of the envelope protein of theMT-2 strain of HTLV-1, in which the alanine (A) residue in position 127,is replaced by a proline (P) residue indicated in bold and underlined,

-   -   polypeptide 5 (SEQ ID NO: 39):

I K K P N R N G G G Y H  S A S Y S D P C S L K C P Y L G C Q S W T C P YA  G A V S S P Y W K F Q Q D V N F T Q E V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 89 to 145 of the envelope protein of theMT-2 strain of HTLV-1, in which the tyrosine (Y) residue in position100, and the threonine (T) residue in position 125, are replacedrespectively by a histidine (H) residue and an alanine (A) residueindicated in bold and underlined,

-   -   polypeptide 6 (SEQ ID NO: 41):

I R  K P N R Q G L G Y Y S P S Y N D P C S L Q C P Y L G S  Q S W T C PY T A  P V S T  P S W N  F H S D V

namely a sequence corresponding to the sequence delimited by the aminoacids situated at positions 85 to 135 of the envelope protein of theprototype NRA strain of HTLV-2, in which the following residues: lysine(K) in position 86, cysteine (C) in position 113, glycine (G) inposition 122, serine (S) in position 126, and lysine (K) in position130, are replaced respectively by the following residues: arginine (R),serine (S), alanine (A), threonine (T), and asparagine (N) indicated inbold and underlined.

A subject of the invention is also the nucleic acids characterized inthat they code for a polypeptide as defined above.

The invention relates more precisely to the abovementioned nucleicacids, comprising the following nucleotide sequences:

-   -   nucleic acid 1 a (SEQ ID NO: 30):

ATT AAA AAG CCA AAC C C A AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which G inposition 281, C in position 302, and G in position 333, are replacedrespectively by C, T, and A indicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the abovementioned polypeptide 1,

-   -   nucleic acid 2 a (SEQ ID NO: 32):

G TT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which A inposition 266, C in position 302, and G in position 333, are replacedrespectively by G, T, and A indicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the abovementioned polypeptide 2,

-   -   nucleic acid 3 a (SEQ ID NO: 34):

ATT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT T T A GCC TCT TAT TCAGAC CCT TGT TCC TTA AA A  TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGCCCC TAT ACA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CA A  CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which C inposition 302, G in position 333, and G in position 408, are replacedrespectively by T, A, and A indicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the polypeptide 3 of claim 24,

-   -   nucleic acid 4 a (SEQ ID NO: 36):

GTT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT TAT TTA GCC TCT TAT TCA GACCCT TGT TCC TTA AAA TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGC CCC TATACA GGA C CC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 417 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which G inposition 379, is replaced by C indicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the abovementioned polypeptide 4,

-   -   nucleic acid 5 a (SEQ ID NO: 38):

ATT AAA AAG CCA AAC CGA AAT GGC GGA GGC TAT C AT TCA GCC TCT TAT TCA GACCCT TGT TCC TTA AAG TGC CCA TAC CTG GGG TGC CAA TCA TGG ACC TGC CCC TATG CA GGA GCC GTC TCC AGC CCC TAC TGG AAG TTT CAG CAA GAT GTC AAT TTT ACC  CA G  GAA GT A

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 265 to 435 of the sequence coding forthe envelope protein of the MT-2 strain of HTLV-1, in which T inposition 298, A in position 373, T in position 426, A in position 429,and T in position 435, are replaced respectively by C, G, C, G, and Aindicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the abovementioned polypeptide 5,

-   -   nucleic acid 6 a (SEQ ID NO: 40):

ATA A GA  AAG CCA AA C  AGA CAG GGC CTA GG G  TAC TAC TCG CC T  TCC TA C AAT GAC CCT TGC TCG CTA CAA TGC CCC TAC TT G  GGC T C C CAA TCA TGG ACATGC CCA TAC ACG G C C CCC GTC TCC A C T CCA TCC TGG AA T  TTT CA T  TCAGAT GTA

namely a sequence corresponding to the sequence delimited by thenucleotides situated at positions 253 to 405 of the sequence coding forthe envelope protein of the NRA strain of HTLV-2, in which A in position257, G in position 258, T in position 267, A in position 282, C inposition 294, T in position 300, A in position 333, G in position 338, Gin position 365, G in position 377, G in position 390, and C in position396, are replaced respectively by G, A, C, G, T, C, G, C, C, C, T, and Tindicated in bold and underlined,

or any nucleotide sequence derived by degeneration of the genetic codeand coding for the abovementioned polypeptide 6.

The invention also relates to the polyclonal or monoclonal antibodiesdirected against a novel variant of type HTLV-1 or HTLV 2 as definedabove, or against a polypeptide defined above, said antibodies being asobtained by immunization of an appropriate animal with an abovementionedpolypeptide.

A subject of the invention is also any pharmaceutical composition, inparticular therapeutic vaccines or vectors, formed from of the novelvariants of type HTLV-1 or HTLV-2 as defined above, and moreparticularly any pharmaceutical composition comprising a polypeptideaccording to the invention as defined above, in particular thepolypeptides 1 to 6 defined above, or a nucleic acid 1a to 6a definedabove, or the abovementioned antibodies, if appropriate in combinationwith a pharmaceutically acceptable vehicle.

The invention also relates to the use of the novel variants of typeHTLV-1 or HTLV-2 as defined above, or the polypeptides according to theinvention as defined above, in particular the polypeptides 1 to 6, orthe nucleic acids 1a to 6a defined above, or the abovementionedantibodies, for the preparation of medicaments intended for theprevention or treatment of infections of an individual by theabovementioned PTLVs, as well as the pathologies defined above linked toinfection by these PTLVs.

The invention is further illustrated by the detailed description whichfollows for obtaining primers according to the invention and of theiruse for the detection of novel variants of HTLV.

I—Development of Molecular Tools and Strategies for the Detection ofPAN-PTLV Sequences by Amplification, Cloning and Sequencing ofNucleotide Sequences Related to the Su of PTLV Envelopes.

1. Screening for Peptide Units Conserved in the N-Terminus of the SU ofPTLV

The main question resolved by the inventors is the development of toolsand of a method allowing the amplification, cloning and identificationof any nucleotide sequence related to the SU of PTLVs which isresponsible for the recognition of their cell receptor (Kim et al.,2000). To this end, the inventors looked for peptide units conserved inthe SU of PTLV envelopes in order to deduce therefrom nucleotidesequences which allow representation of them all. These peptide unitsshould ideally meet the following 5 criteria, in decreasing order ofimportance:

-   -   To be conserved in most, if not all, of the PTLV envelope        sequences already described. Such a conservation would be a        guarantee of their potential effectiveness in the detection of        new sequences of PTLV type.    -   To represent at least 5 conserved amino acids from the SU of        PTLVs, in order to derive therefrom a minimum sequence of 15        nucleotides. Given the complexity of the eukaryotic genomes,        this minimum of 15 nucleotides is required to allow the specific        detection of a given nucleotide sequence.    -   To allow the amplification of sequences situated upstream of the        C I/M V C unit which is conserved in the SU of PTLVs and seems        to be analogous to the CWLC unit described in the SU of MuLVs        (Sitbon et al., 1991). This unit seems in fact, to mark a hinge        region between, upstream of it, the part of the SU responsible        for the recognition of the receptor and, downstream of it, the        carboxyl-terminal domains of the SU involved in the association        with the TM and stages of viral entry subsequent to the        recognition of the receptor (Battini, et al., 1992; Battini et        al., 1995; Lavillette et al., 1998; Kim et al., 2000; Lavillette        et al., 2001). To be sufficiently distant from each other to        allow the amplification of a fragment whose length would        increase the chances of detection of a possible polymorphism        between different sequences.    -   To be situated so as to allow two successive DNA amplification        reactions, the second of which, nested, is produced from        products of the first amplification, and allows the        amplification of a fragment internal to the first fragment        amplified. This nested amplification allows an increase in the        probability of amplification of a fragment which corresponds        well to a sequence related to the SU of PTLVs.

According to these criteria, the inventors have identified the followingamino acid units, present in all or almost all of the known SUs of PTLV,and being able to help in the development of this strategy:

-   -   Peptide unit 1: Y L/V F P H W    -   Peptide unit 2: N F T Q/R E V    -   Peptide unit 3: N Y S/T C I/M V C    -   Peptide unit 4: W H V L Y

2. Degenerated Synthesis Oligonucleotides Corresponding to the UnitsConserved in the Amino-terminal Part of the SU of PTLVs

Using amino acid sequences of the conserved peptide units identifiedabove and following the nucleotide correspondence in the application ofthe eukaryotic genetic code, the inventors determined degeneratednucleotide sequences (DNS) which served as the basis for the design ofsynthesis oligonucleotides (SO). Several criteria governed the design ofSO corresponding to these DNS:

-   -   When the multiplication of the degenerated positions in a DNS        would make the complexity of the corresponding SO exceed 512        oligonucleotides in the synthesis mixture, the synthesis of        supplementary SO for this DNS is then carried out to remove some        of this complexity.    -   The synthesis of one or 2 supplementary OS, to a limit of 4 SO        per DNS, is carried out even for complexities less than 512,        when these supplementary SO significantly remove the complexity        of the initial degenerated OS.    -   The sequences of the 5′ SO, the extension of which by the DNA        polymerases must correspond to the amino acids situated upstream        of the peptide unit considered (units 1 and 2), are those of the        DNA (+) strand, while those of the 3′ SO, the extension of which        must correspond to the amino acids situated upstream of the        peptide unit considered (units 2, 3 and 4), are those of the DNA        (−) strand. The SO corresponding to the peptide unit 2 were        synthesized on the two strands, in order to be able to carry out        an extension in both directions.    -   Each SO comprises supplementary nucleotides allowing the        introduction at 5′ of the sequence corresponding to a        restriction site, EcoRI for the 5′ OS, BamHI for the 3′ SO, and        in all the cases a GGAA 5′-terminal sequence favouring the        binding of the polymerases and nucleases upstream of the        restriction site.    -   According to these criteria, the SO PTLVE5′ (83 a and b, 140 a        to d) and PTLVE3′ (145 a to d, 228 a to h, 241 a and b) (for        Primate T-Leukemia Virus-like Env), defined above were        synthesized respectively for elongations at 5′ or 3′ of the        targeted unit.

3. Development of the Amplification Conditions with Oligonucleotides onControl Sequences

For the development of the amplification of sequences recognized by theSO described above, the inventors used control plasmid DNA preparationscontaining the HTLV-1 sequence envelope and control preparations nothaving this sequence. The DNA amplification strategy chosen consists oflinking two amplification reactions by a mixture of the Taq and Pwopolymerases on a thermal cycler under conditions called “touch-down” andcombining 2 different SO pairs.

The first probative and reproducible amplification results (specificamplification of HTLV sequences without amplification on the controlpreparations) are those obtained with the combination of the SOPTLVE5′83b and PTLVE3′240b, for the first amplification reaction,followed by a 2^(nd) reaction combining the SO PTLVE5′83b andPTLVE3′146a on a sample of the 1^(st) reaction. In both cases the“touch-down” conditions include 15 cycles combining each denaturation at94° C. followed by a stage of annealing and extension carried out at thesame temperature, this temperature being comprised for each cyclebetween 65 and 50° C. with a decreasing step of 1° C. between the 1^(st)and the 15^(th) cycle. These 15 cycles are followed by 30 standardamplification cycles with an annealing temperature at 50° C. and anextension temperature at 72° C.

4. Construction and Sequencing of a Bank of Fragments Amplified fromAmplification Reactions

A sample of the 2^(nd) amplification reaction described above is used togenerate a bank of the amplified sequences. To do this 4 μl of the 50 μlof the 2^(nd) reaction is used for ligation in a pCR4-TOPO type vector(Invitrogen) and transformation of bacteria. Between 10 and 100 coloniesresistant to kanamycin are subcultured for each ligation and placed inculture. The plasmid DNA of each colony is analysed by sequencing usinguniversal primer sequences T3 and T7 of the vector.

II—First Results Obtained from Human and Primate Samples

The conditions described above were applied to three types of DNAsamples:

-   -   Samples of genomic DNA of “seroindeterminate patients”,        characterized by a serology suggesting a previous infection with        HTLV but in whom no definitive diagnosis was able to be        established. In these patients, in particular, research by DNA        amplification of gag, pol or tax HT-LV sequences is negative.    -   Samples of genomic DNA of “HTLV-1 patients” in whom a        characteristic HTLV-1 infection was identified.    -   Samples of genomic DNA of Agile Mangabey monkeys (Cercocebus        Agilis) which have a positive PTLV serology and in which Tax        HTLV-1 or STLV-L sequences were able to be amplified.

The application of the method described above allowed detection of thepresence of SU type PTLV sequences in the three types of samples,including in the “seroindeterminate patients”.

Analysis of the sequences and of their coding capacities at the level ofthe SU region concerned allowed the following observations to be made:

1. Results Obtained on “Seroindeterminate Patients”

By applying the method described above on the DNA of a“seroindeterminate patient” (sample No. 424), described as not having anHTLV type sequence, the inventors were however able to amplify andcharacterize SU type PTLV sequences.

At the nucleotide level, the sequences identified from sample No. 424are of several types: HTLV-1 sequences identical to those alreadydescribed in the literature and new variants. At the coding level, thenucleotide sequences translate into three types of sequences:

-   -   Amino acids sequences identical to those of the HTLV-1 strains        already known.    -   Variants of HTLV-1 strains with 1 or 2 residues not described        previously.    -   Variants of HTLV-1 strains with 1 or 2 residues described as        common only to the HTLV-2 or STLV-L strains.

2. Results Obtained on “Typical HTLV-1 Patients”

At the nucleotide level, the amplified sequences from the sampleoriginating from the “HTLV-1 patient” (sample No. 422) are eithertypically HTLV-1, as already described in the literature, or variantswith repercussions for the coding capacity. At the coding level, thenucleotide sequences translate into three types of sequences:

-   -   Amino acids sequences identical to the known HTLV-1 strains.    -   HTLV-1 variants with 1 or 2 residues typical of HTLV-2, combined        or not combined with residues never previously described.    -   HTLV-2 variants combining a few residues described as being        common only to the HTLV-2 or STLV-L strains, this being combined        or not combined with residues never previously described.

3. Results obtained on Cercocebus Agilis monkeys

The method of the invention also allowed amplification of the SU typePTLV sequences in all the Agile Mangabey monkeys (Cercocebus Agilis)tested which were identified as seropositive for PTLV. At the nucleotidelevel, the sequences amplified from these monkeys are either those ofthe isolates already described previously, or nucleotide variants withrepercussions for the coding capacity. At the coding level, thenucleotide sequences translate into three types of sequences:

-   -   Amino acids sequences identical to the known HTLV-1 strains.    -   Amino acids sequences identical to the isolate STLV-3/CTO-604        recently described in a red-capped Mangabey (Cercocebus        Torquatus) (Meertens et al., 2002)    -   Amino acid sequences of the STLV-3/CTO-604 type with 1 or 2        residues typical of HTLV-2

III—BIBLIOGRAPHY

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2. Battini, J. L., J. M. Heard, and O. Danos. 1992. Receptor choicedeterminants in the envelope glycoproteins of amphotropic, xenotropic,and polytropic murine leukaemia viruses. J. Virol. 66(3):1468-75.

3. Kim, F. J., I. Seiliez, C. Denesvre, D. Lavillette, F. L. Cosset, andM. Sitbon. 2000. Definition of an amino-terminal domain of the humanT-cell leukaemia virus type 1 envelope surface unit that extends thefusogenic range of an ecotropic murine leukaemia virus. J Biol. Chem.275(31):23417-20.

4. Lavillette, D., M. Maurice, C. Roche, S. J. Russell, M. Sitbon, andF. L. Cosset. 1998. A proline-rich motif downstream of the receptorbinding domain modulates conformation and fusogenicity of murineretroviral envelopes. J. Virol. 72(12):9955-65.

5. Lavillette, D., A. Ruggieri, S. J. Russell, and F. L. Cosset. 2000.Activation of a cell entry pathway common to type C mammalianretroviruses by soluble envelope fragments. J. Virol. 74(1):295-304.

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1. A method for implementing processes for detecting a strain of PTLV,said process comprising the steps of: (a) amplifying a target nucleicacid in a biological sample capable of containing PTLVs with pairs ofdegenerate 5′ and 3′ oligonucleotides in the 5′ and 3′ orientationoriginating from the nucleotide sequences coding for the amino-terminalregion of the surface component (SU) of the envelope proteins of theviruses of T lymphomas/leukaemias in primates, collected together underthe designation PTLV, said viruses being designated HTLV in man and STLVin the monkey, wherein the amino-terminal region is a protein fragmentdelimited on the N-terminal side by an amino acid situated betweenpositions 75 to 90, and on the C-terminal side by an amino acid situatedbetween positions 130 to 145 of the envelope protein of the MT-2 strainof HTLV-1 represented by SEQ ID No: 43, or of the envelope protein ofthe NRA strain of RTLV-2 represented by SEQ ID NO: 45, or of theenvelope protein of the strain of STLV-3 represented by SEQ ID NO: 47,or a virus carrying the sequences belonging to the SU of PTLVs, and (b)identifying the strain of PTLV contained in the biological sample bydetermining nucleotide fragments that have been amplified.
 2. The methodaccording to claim 1, wherein said strain of PTLV is selected from thegroup consisting of HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3.
 3. Themethod according to claim 1, wherein said degenerate oligonucleotidesare chosen from those comprising approximately 15 to approximately 30nucleotides originating from the nucleotide sequences coding for proteinfragments delimited on the N-terminal side by an amino acid situatedbetween positions 75 to 90, and on the C-terminal side by an amino acidsituated between positions 130 to 145 of the envelope protein of theMT-2 strain of HTLV-1 represented by SEQ ID NO: 43, or of the envelopeprotein of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or ofthe envelope protein of the strain of STLV-3 represented by SEQ ID NO:47, and wherein said degenerate oligonucleotides comprise a mixture ofoligonucleotides originating from sequences coding for a determinedregion of approximately 5 to 10 amino acids of the envelope proteins ofthe different strains of PTLV, and which differ from each other by thesubstitution of at least one nucleotide by another in a manner such thateach oligonucleotide is capable of coding for the abovementioneddetermined region originating from the protein fragments of the envelopeproteins of the different strains of PTLVs.
 4. The method according toclaim 1, wherein pairs of degenerate oligonucleotides compriseapproximately 15 to approximately 30 nucleotides originating fromnucleotide sequences coding for polypeptide fragments of approximately 5to approximately 10 amino acids originating from protein fragmentsdelimited by the amino acids situated at positions 80 to 145, and moreparticularly at positions 83 to 145, of the envelope protein of the MT-2strain of HTLV-1 represented by SEQ ID NO:
 43. 5. The method accordingto claim 1, wherein the pairs of degenerate oligonucleotides originatefrom nucleotide sequences coding for the polypeptide fragment 83-88represented by SEQ ID NO: 1, and polypeptide fragment 140-145represented by SEQ ID NO: 2, of the envelope protein of the MT-2 strainof HTLV-1
 6. The method according to claim 1, wherein the degenerateoligonucleotides are degenerate oligonucleoticles in 5′ orientationoriginating from the DNA (+) strand coding for a polypeptide selectedfrom the group consisting of: a polypeptide fragment 83-88 of theenvelope protein of the MT-2 strain of HTLV-1, said oligonucleotidesbeing chosen from those of following formula (I): . TAYBTNTTYCCNCAYTGG(I) SEQ ID NO: 5

in which: Y represents C or T, B represents C, G or T, N represents A,C, G or T, provided that when N represents T, B cannot represent T, anda polypeptide fragment 140-145 of the envelope protein of the MT-2strain of HTLV-1, said oligonucleotides being chosen from those offollowing formula (II): AAYTTYACNCARGARGT (II) SEQ ID NO: 8

in which: Y represents C or T, R represents A or G, N represents A. C, Gor T.
 7. The method according to claim 5, wherein the degenerateoligonucleotides are degenerate oligonucleotides in 3′ orientationoriginating from the DNA (−) strand coding for the polypeptide fragment140-145 of the envelope protein of the MT-2 strain of HTLV-1, andwherein said oligonucleotides are of formula (III): NACYTCYTGNGTRAARTT(III) SEQ ID NO: 13

in which: Y represents C or T, R represents A or G, N represents A, C, Cor t.
 8. The method according to claim 1, wherein the pairs ofdegenerate oligonucleotides are selected from the group consisting of: adegenerate 5′ oligonucleotide corresponding to a mixture of 5′oligonucleotides originating from a same determined nucleotide regioncomprising approximately 15 to approximately 30 nucleotides originatingfrom the DNA (+) strand and coding for the polypeptide fragments ofapproximately 5 to approximately 10 amino acids originating from proteinfragments delimited on the N-terminal side by an amino acid situatedbetween positions 75 to 90, and on the C-terminal side by an amino acidsituated between positions 135 to 150 of the envelope protein of theMT-2 strain of HTLV-1 represented by SEQ ID No: 43, or of the envelopeprotein of the NRA strain of HTLV-2 represented by SEQ ID NO: 45, or ofthe envelope protein of the strain of STLV-3 represented by SEQ ID NO:47, said 5′ oligonucleotides are different from each other bysubstitution of at least one nucleotide by another such that eacholigonucleotide is capable of coding for the abovementioned determinedregion originating from protein fragments of the envelope proteins ofdifferent strains of PTLVs, and a degenerate 3′ oligonucleotidecorresponding to a mixture of 3′ oligonucleotides originating from asame determined nucleotide region comprising approximately 15 toapproximately 30 nucleotides originating from the DNA (−) strand andcoding for the polypeptide fragments of approximately 5 to approximately10 amino acids originating from protein fragments delimited on theN-terminal side by an amino acid situated between positions 125 to 145,and on the C-terminal side by an amino acid situated between positions130 to 145 of the envelope protein of the MT-2 strain of HTLV-1represented by SEQ ID No: 43, or of the envelope protein of the NRAstrain of HTLV-2 represented by SEQ ID NO: 45, or of the envelopeprotein of the strain of STLV-3 represented by SEQ ID NO: 47, whereinsaid 3′ oligonucleotides being such that they differ from each other bysubstitution of at least one nucleotide by another such that eacholigonucleotide is capable of coding for the abovementioned determinedregion originating from the protein fragments of the envelope proteinsof the different strains of PTLVs, and wherein said 5′ and 3′ primerscannot be complementary to each other.
 9. The method according to claim7, wherein the degenerate 5′ oligonucleotides are selected from thegroup consisting of SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:13.
 10. Themethod according to claim 7, wherein the degenerate 5′oligonucleotides_encode for the protein fragments comprising a sequencedelimited on the N-terminal side by the amino acid situated in position89, and on the C-terminal side by the amino acid situated in position139 of the envelope protein of the MT-2 strain of HTLV-1 represented bySEQ ID NO: 43, or a sequence comprising an envelope protein of a strainof PTLV other than HTLV-1, wherein the sequence delimited on the N-terminal side by the amino acid situated in position 85, and on theC-terminal side by the amino acid situated in position 135 of theenvelope protein of the NRA strain of HTLV-2 represented by SEQ ID NO:45, or a sequence delimited on the N-terminal side by the amino acidsituated in position 88, and on the C-terminal side by the amino acidsituated in position 144 of the envelope protein of the strain of STLV-3represented by SEQ ID NO:47.
 11. The method according to claim 10,wherein the degenerate 5′ oligonucleotide is SEQ ID NO:5 or SEQ IDNO:13.
 12. The method according to claim 10, wherein the degenerate 5′oligonucleotides are SEQ ID NO: 5 and SEQ ID NO:
 14. 13. A process fordetecting any strain of PTLV, as well as any strain of virus comprisingthe sequences belonging to the SU of PTLVs, said process comprising:contacting a pair of degenerate 5′ and 3′ oligonucleotides encoding forpeptides of SEQ ID NO: 1 and SEQ ID NO: 2 with genomic DNA orcomplementary DNA derived from a biological sample containing PTLV,amplifying DNA fragments of said genomic DNA or complementary DNAderived from a biological sample containing PTLV coding for a fragmentof the envelope proteins of the different strains of PTLVs, anddetecting the DNA fragments amplified during the previous stage todetermine whether said sample contains said PTLV.
 14. The process ofclaim 13, wherein said strains of PTLVS are selected from the groupconsisting of: HTLV-1, HTLV-2, STLV-1, STLV-2, and STLV-3.
 15. A processfor detecting a strain of PTLV comprising SEQ ID NO: 43, said processcomprising: contacting genomic DNA or complementary DNA derived from abiological sample containing said PTLV with a pair of degenerate 5′ and3′ oligonucleotides encoding for peptides of SEQ ID NO: 1 and SEQ ID NO:2, amplifying DNA fragments of said genomic DNA or complementary DNAderived from a biological sample containing PTLV, and detecting the DNAfragments amplified during the previous stage to determine whether saidsample contains said PTLV.